Simple Synthesis of Hierarchically Ordered Mesocellular Mesoporous Silica Materials Hosting Crosslinked Enzyme Aggregates

Porous materials

Simple Synthesis of Hierarchically OrderedMesocellular Mesoporous Silica Materials HostingCrosslinked Enzyme AggregatesJinwoo Lee, Jungbae Kim,* Jaeyun Kim, Hongfei Jia, Moon Il Kim, Ja Hun Kwak, Sunmi Jin,Alice Dohnalkova, Hyun Gyu Park, Ho Nam Chang, Ping Wang, Jay W. Grate, and Taeghwan Hyeon*

Hierarchically ordered mesocellular mesoporous silica materials(HMMS) were synthesized using a single structure-directing agent. Themesocellular pores are synthesized without adding any pore expander;the pore walls are composed of SBA-15 type mesopores. Small-angle X-ray scattering revealed the presence of uniform pore structures with twodifferent sizes. Using HMMS as a nanoscopic template, hierarchicallyordered mesocellular mesoporous carbon (HMMC) and polymer(HMMP) materials were synthesized. HMMS was used as a host forenzyme immobilization. To improve the retention of enzymes in HMMS,we adsorbed enzymes, and then employed crosslinking using glutar-aldehyde (GA). The resulting crosslinked enzyme aggregates (CLEAs)show an impressive stability with extremely high enzyme loadings. Forexample, 0.5 g a-chymotrypsin (CT) could be loaded in 1 g of silica withno activity decrease observed with rigorous shaking over one month. Incontrast, adsorbed CT without GA treatment resulted in a lower loading,which further decreased due to continuous leaching of adsorbed CTunder shaking. The activity of crosslinked CT aggregates in HMMS was�10 times higher than that of the adsorbed CT, which represents a 74-fold increase in activity per unit weight of HMMS due to higher CTloading.

Keywords:· crosslinking· enzymes· mesoporous materials· silica· template synthesis

[*] Dr. J. Kim, Dr. J. H. Kwak, A. Dohnalkova, Dr. J. W. GratePacific Northwest National LaboratoryRichland, WA 99352 (USA)Fax: (+1) 509-375-5106E-mail: [email protected]

Dr. J. Lee, J. Kim, S. Jin, Prof. Dr. T. HyeonNational Creative Research Initiative Center for OxideNanocrystalline Materials andSchool of Chemical EngineeringSeoul National University, Seoul 151-744 (Korea)Fax: (+82) 2-888-1604E-mail: [email protected]

H. Jia, Prof. Dr. P. WangDepartment of Chemical EngineeringUniversity of Akron, Akron, Ohio 44325 (USA)

M. I. Kim, Prof. H. G. Park, Prof. H. N. ChangDepartment of Chemical and Biomolecular EngineeringKorea Advanced Institute of Science and Technology, Daejeon305-701 (Korea)

Supporting information for this article is available on the WWWunder http://www.small-journal.com or from the author.

744 � 2005 Wiley-VCH Verlag GmbH & Co. KGaA, D-69451 Weinheim DOI: 10.1002/smll.200500035 small 2005, 1, No. 7, 744 –753

full papers J. Kim, T. Hyeon, et al.

1. Introduction

Mesoporous materials with pore sizes ranging from 2 to50 nm have attracted considerable attention because of theirmany applications involving large molecules, which cannotbe accomplished using conventional microporous zeoliticmaterials.[1] Amphiphilic block copolymers, along with sur-factant self-assemblies, have proven to be valuable supra-molecular templates for the synthesis of ordered mesostruc-tured materials.[2] Mesocellular siliceous foam (MCF) withultra-large mesocellular pores connected by mesoporouswindows have been fabricated using tri-block copolymers asa structure-directing agent, and 1,3,5-trimethylbenzene(TMB) as a pore expander.[3] Complementary pores, formedby the penetration of ethylene oxide (EO) groups, are pres-ent in the walls of the MCF.[4] However, these complemen-tary pores are disordered micropores smaller than 2 nm. Re-cently, active research has been conducted on the synthesisof hierarchically ordered porous materials.[5] Generally, dualtemplates were used to synthesize such materials. Polystyr-ene (PS) beads have frequently been used for the genera-tion of macropores, while smaller-sized templates were usedfor the mesopores. In a previous study, macroporous silicamaterials with zeolitic walls were synthesized using PSbeads and tetrapropyl ammonium hydroxide (TPAOH) asdual templates.[5a] Preformed silicalite nanoparticles andstarch gel were used to make macroporous/microporous ma-terials.[5b] Macro/mesoporous silica has been produced usingPS beads and low-molecular-weight surfactants or amphi-philic triblock copolymers as dual templates.[5c,d] Monolithicsilica[5e] and silica films[5f] with bimodal pore structures werealso prepared. Bimodal super-microporous and macropo-rous silica material was synthesized using a PS bead packingand an ionic liquid as dual templates.[5h] Trimodal poroussilica was prepared using PS beads for macropores, a blockcopolymer for large mesopores, and an ionic liquid for smallmespores, respectively.[5i] Recently, bimodal porous materi-

als were synthesized using a single template. Antonelli re-ported the synthesis of macro/mesoporous niobium oxidemolecular sieves by adding a large amount of NaCl andusing a single dodecylamine surfactant.[6] The author claim-ed that NaCl played a crucial role in the formation of thelarge-sized vesicles that led to the generation of the macro-pores. More recently, bimodal porous materials of titaniaand zirconia with wormhole-like mesopores and a funnel-like macrostructure were synthesized using single-surfactantdecaoxyethylene cetyl ether (C16(EO)10).[7] The generationof the macropores was attributed to the vesicle-type super-micelles formed by the aggregation of unreacted excess sur-factants.

Herein, we report on the synthesis of a mesocellularsilica foam with ordered mesoporous walls, designated as hi-erarchical mesocellular mesoporous silica (HMMS), usingan amphiphilic triblock copolymer as a single structure-di-recting agent. The synthetic method for HMMS is verysimple and cost-effective. Generally, to make mesocellular-type pores, a pore expander[3] such as trimethylbenzene isrequired. However, in this synthesis, the mesocellular poresare synthesized without adding an additional pore expander.Interestingly, the walls of the cellular pores in HMMS arecomposed of SBA-15-type pores. The final pore structure ofHMMS is a mixed structure of mesocellular silica foam(MCF)[3] pores and one-dimensional SBA-15 pores.[2a] UsingHMMS silica as a template, hierarchical mesocellular meso-porous carbon and polymer materials were successfully fab-ricated.

As a possible application of HMMS, we attempted toimmobilize enzymes in HMMS. Mesoporous materials haveattracted much attention for enzyme immobilization,[8,9] andmuch effort to improve both enzyme loading and stabilityhas been made.[9] In this paper, we have developed cross-linked enzyme aggregates (CLEAs)[10] in HMMS, which em-ploys the adsorption of enzymes followed by enzyme cross-linking using glutaraldehyde. Since the multipoint attach-ment of enzyme molecules is well-known to stabilize activityby preventing enzyme denaturation,[11] we anticipated thatCLEAs in HMMS would stabilize enzyme activity. Duringthe measurement of enzyme stability, all immobilized en-zymes were incubated under rigorous shaking. Even thoughshaking is required in a conventional enzyme reactor usingimmobilized enzymes, few reports have analyzed the longev-ity of immobilized enzymes in mesoporous materials undershaking. This unprecedented approach (using shaking) waspossible because CLEAs in HMMS clearly stabilized theenzyme activity.

2. Results and Discussion

2.1. Synthesis and Characterization of HMMS

The structure of HMMS is presented in Figure 1. Meso-cellular siliceous foam (MCF)[3] is composed of large cells

Editorial Advisory Board Member

Taeghwan Hyeon received his BS (1987)and MS (1989) in Chemistry from SeoulNational University, Korea. He obtainedhis PhD from the University of Illinois atUrbana-Champaign (1996). Since hejoined the faculty of the School of Chem-ical and Biological Engineering of SeoulNational University in 1997, he has beenfocused on the synthesis of uniform-sized nanocrystals and new nanoporouscarbon materials. He is currently Directorof the National Creative Research Initia-

tive Center for Oxide Nanocrystalline Materials. Over the past fiveyears, he has published more than 70 papers in prominent interna-tional journals. He has received several awards, including the T. S.Piper Award from the University of Illinois, the Korean Young Scien-tist Award from the Korean President, and the Dupont ScientistAward.

small 2005, 1, No. 7, 744 –753 www.small-journal.com � 2005 Wiley-VCH Verlag GmbH & Co. KGaA, D-69451 Weinheim 745

Hierarchical Mesoporous Materials

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(>20 nm) and connecting windows. To make MCF, oil-in-water microemulsions were used as structure-directingagents for structure assembly. Hexagonally ordered mesopo-rous silica[2a] is synthesized using P123 ((EO)20(PO)70(EO)20)as a structure-directing agent. The addition of trimethylben-zene (TMB) to a P123 solution as a pore expander convertshexagonally ordered structures to mesocellular structures,which exhibit spherical pores.[3b] HMMS has two interestingpore structures in one mesostructured primary particle. Inthis synthesis, two pore types are formed using only oneP123 template. MCF-type cellular pores are surrounded bySBA-15-type mesopores. To our knowledge, there havebeen no reports on this type of hierarchical mesoporous–mesoporous material. Most hierarchical porous materialsare macro–meso- or macro–microporous materials.

HMMS was characterized by transmission electron mi-croscopy (TEM), scanning electron microscopy (SEM), gasadsorption measurements, and small-angle X-ray scattering.A representative transmission electron microscopic imageof HMMS (Figure 2a) shows that �10-nm-sized orderedmesopores are associated with larger �40-nm-sized meso-cellular pores. The cellular pores in HMMS are clearlyshown in the scanning electron microscopic image (Fig-ure 2b). The TEM image (Figure 2c), obtained by thin-sec-tioning a polymer-embedded sample, clearly shows that thelarge cellular pores of the HMMS are well-mixed with thesmaller �10-nm-sized ordered mesopores. The structure ofHMMS is a mixture of MCF[3] and SBA-15 silica[2a] in onemesostructured primary particle. Representative nitrogenadsorption/desorption isotherms and the correspondingpore-size distribution obtained from the analysis of the ad-sorption branch using the BJH (Barett–Joyner–Halenda)method are shown in Figure 3a. The nitrogen isotherm

shows two major capillary condensation steps at relativelyhigh pressures of over 0.8 P/P0. The step at 0.8–0.9 P/P0 re-sults from adsorption in the �10-nm-sized mesopores, whilethe other at 0.9–1.0 P/P0 is from adsorption in the �40-nm-sized cellular mesopores. The presence of these two distincttypes of pores is also clearly revealed in the pore-size distri-bution (PSD), which shows two peaks centered at 13.3 nmand 36.6 nm derived from the main mesopores and mesocel-lular pores, respectively. The BET surface area and single-point total pore volume of HMMS are 330 m2 g�1 and1.34 cm3 g�1, respectively. The micropore volume, obtainedby the Horvath–Kawazoe method based on a low-pressureisotherm, was about 10 % of the total pore volume of theHMMS silica. These micropores seem to have been generat-ed as a result of the penetration of ethylene oxide (EO)groups into the silica walls.[4] From these characterizations,it is evident that HMMS is composed of 37 nm cellular mes-opores and 13 nm ordered mesopores.

Generally, only small mesopores (<10 nm) exhibit anordered structure in hierarchical mesostructured materials.

Figure 1. Cartoon showing that the structure of HMMS is a mixedstructure of mesocellular type pores and one-dimensional-channelSBA-15-type pores.

Figure 2. a) Representative transmission electron microscopic (TEM)image of HMMS. b) Representative scanning electron microscopic(SEM) image of HMMS. c) TEM image of HMMS after microtoming.

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Surprisingly, however, the small-angle X-ray scattering(SAXS) pattern of HMMS revealed the presence of orderedpore structures with two different length scales. Figure 3bshows the SAXS spectrum obtained from HMMS, whichshows two sets of scattering peaks that are indicated by thetwo closed circles at q=0.21 and 0.42 nm�1, and two aster-isks at q=0.55 and 0.97 nm�1, where the first two peaks cor-respond to the reflections from the �30 nm pores, and thelatter two peaks derive from the hexagonal (100) plane withan interplanar spacing (d) of 11.3 nm. The calculated valuesof the interplanar spacing (d100) and lattice parameter (a)from the small mesopores with a hexagonal phase are about11.3 nm and 13 nm, respectively, while the larger cellularmesopores have a size of 31 nm. These results clearly dem-onstrate that the two different sets of pores are very uni-form in size. When P123 was used as a structure-directingagent for assembling the mesostructure, only unimodalpores are generally obtained.[2a] To make mesocellular typepores, a pore expander such as trimethylbenzene is usuallyrequired.[3] However, in the synthesis of HMMS, mesocellu-lar pores were generated without adding any pore expander.

A plausible synthetic route for HMMS is schematicallyshown in Figure 4. In our synthesis, sodium silicate solution

is diluted with a large amount of water. When we used con-centrated sodium silica instead of diluted sodium silicate,we obtained poorly defined silica materials. The pH value ofboth the undiluted and diluted sodium silicate solutions isover 14. Above pH 12, most silanols are known to be depro-tonated and the major silica building blocks are present inthe form of cyclic trimers and tetramers.[12] The interactionbetween P123 surfactant and the silica source is mainlybased on hydrogen bonding under neutral conditions. Thediluted sodium silicate can be neutralized faster than con-centrated sodium silicate by acetic acid present in the syn-thetic medium. Thus the diluted sodium silicate interactswith the tri-block copolymers faster, resulting in the produc-tion of small-sized mesostructured building units. The resid-ual P123 would be expected to interact with these pre-formed units, resulting in the formation of bilayer supermi-celle structures.[7a,13] The continuous cooperative synthesis ofbilayer or globular structures on a silica surface was report-ed by Grant et al,[13] while Su and co-workers reported onhierarchical macro–mesoporous transition-metal oxide ma-terials.[7a] They also used a single surfactant to make dualmacro–mesoporous materials. The formation of hierarchicalmacro–mesoporous materials was explained by fast hydroly-sis/condensation of transition metal alkoxides resulting inthe formation of small mesostructured building units with alarge number of hydroxyl groups, and the assembly of resid-ual surfactants into multilayered micelles to make macro-pores. In our synthesis, prehydrolyzed sodium silicate wasused instead of tetraethyl orthosilicate (TEOS). The pH val-ue of our synthetic medium is 6.3–6.4 and the condensationrate is a maximum at around pH 6.[12] So, through a similarpathway, small-sized mesostructure silica building blockswith SBA-15 pores templated by P123 can be initiallyformed. Residual surfactants first adsorb onto the hydro-philic surface and self- assemble into large vesicle-type mi-celles, which are responsible for the formation of the meso-cellular pores.

In our study, the formation of a hierarchically orderedmesocellular mesoporous structure before hydrothermaltreatment is corroborated by TEM and nitrogen isotherms(see Supporting Information). Hydrothermal treatment wasconducted to make the silica walls more rigid. To demon-strate that the residual P123 surfactant plays a key role as a

Figure 3. a) N2 adsorption/desorption isotherms of HMMS (Inset: Thecorresponding pore-size distribution). b) Small-angle X-ray scatteringpattern of HMMS showing the regularity of large cellular pores andsmall ordered mesopores.

Figure 4. Schematic representation showing the formation mecha-nism of HMMS.




structure-directing agent for large cellular pores, the amountof P123 surfactant was varied, while keeping the other ex-perimental conditions unchanged. When the amount ofP123 was increased by a factor of 1.1–1.3, HMMS with a hi-erarchical structure was obtained (Figure 5a). However,

when the amount of P123 was reduced to only 0.7 times thestandard amount, one-dimensional, hexagonally orderedsilica with unimodal pores, similar to the SBA-15 structure,was obtained (Figure 5b). Pinnavaia and co-workers report-ed the synthesis of ordered one-dimensional mesoporousmolecular sieves (pore size �10 nm), denoted as MSU-H,[14]

under near-neutral synthetic conditions. Although the syn-thetic method used for the fabrication of the HMMS is simi-lar to that of MSU-H, HMMS possesses uniformly sized cel-lular mesopores along with �13-nm-sized mesopores, where-as no such cellular mesopores were observed in MSU-H.

2.2. Hierarchical Mesocellular Mesoporous Carbon andPolymer Materials from a HMMS Silica Template

Uniform-sized mesoporous carbon materials formedusing various mesostructured silica templates have attractedmuch attention for their possible application as electrodematerials, adsorbents, and catalyst supports.[15] Recently, ourgroup synthesized two kinds of bimodal mesoporous car-bons using MCF, developed by Stucky and co-workers, andbimodal mesoporous silica, developed by our group, as inor-ganic templates.[16,17] The key to the success of the prepara-tion of these bimodal mesoporous carbons is the selectiveincorporation of carbon precursors. In the synthesis of mes-ocellular carbon foam, phenol vapor could not infiltrate intothe large main cellular mesopores because a very highvapor pressure was required. Using a similar synthetic pro-cedure and employing HMMS as the silica template, howev-er, we were able to synthesize a new hierarchical mesocellu-lar mesoporous carbon (HMMC). Although the synthesis ofthe mesocellular carbon foam with a bimodal pore structurehas already been reported by our group, the small meso-pores present in the walls were disordered pores with sizes

of 3–4 nm.[16] In contrast, the current HMMC exhibited or-dered mesopores along with the main cellular pores. Theoverall synthetic scheme for HMMC is presented in theSupporting Information.

The TEM image of HMMC (Figure 6a) shows that the�40 nm cellular pores of HMMS are well preserved andthat small mesopores are also present. These small meso-

pores were generated by the replication of the 13 nm meso-pores of the HMMS silica. The preservation of the large cel-lular pores is also confirmed by SEM (Figure 6b). The indi-vidual particle size of HMMC is also a few hundred nano-meters, which is favorable for the adsorption of large mole-cules when used as an adsorbent or as a nanometer-scalereactor for biomolecules.[16b]

The N2 isotherms of HMMC (Figure 7a) exhibited twomajor capillary condensation steps, resulting from the largecellular pores (P/P0�0.9) and small ordered mesopores(P/P0�0.6), respectively. The preservation of the orderedstructure of the small pores contained in HMMS is attribut-ed to the presence of the complementary pores present be-tween the ordered small pores (�13 nm).[4] The size of thepores generated from the dissolution of the silicate walls is4.74 nm, as calculated using the adsorption isotherm basedon the BJH method, which is somewhat larger than that ofCMK-3[18] or C-MSU-H.[19] The pore-size distribution result-ing from the large cellular pores (�40 nm) is somewhatbroadened compared with that of HMMS. The BET surfacearea and single-point total pore volume of HMMC are

Figure 5. TEM images of mesoporous silica synthesized by usingdifferent amounts of P123 from the original recipe. a) When theamount of P123 was increased by 1.3 times, the resulting silicaexhibited a hierarchical dual mesoporous structure consisting oflarge mesocellular pores and small mesopores. b) Reducing theamount of P123 to 0.7 times the original amount resulted in aunimodal silica material with a one-dimensional structure.

Figure 6. a) Representative TEM image of HMMC. b) RepresentativeSEM image of HMMC.




853 m2 g�1 and 1.54 cm3 g�1, respectively. The SAXS patternof HMMC revealed that the regularity of both the large cel-lular pores and small mesopores of HMMS is preservedduring the replication (Figure 7b). The SAXS pattern showstwo sets of scattering peaks. The peak indicated by thesingle closed circle results from the large mesocellularpores, while those indicated by the four asterisks are fromthe small ordered mesopores. The calculated values of theinterplanar spacing (d100) and lattice parameter (a) of thesmall mesopores are about 10.0 nm and 11.7 nm, respective-ly. The peak indicated by the closed circle in Figure 7b (q=

0.18 ��1) represents the mesocellular pores have a pore sizeof about 35 nm.

Mesoporous polymers with interconnected large meso-pores and high surface areas play a very important role assolid-phase supports for organic reactions that require bulkyreagents.[20] A mesocellular mesoporous polymer foam com-posed of poly(divinylbenzene) with hierarchically orderedmesopores, here denoted as HMMP, was successfully syn-thesized using HMMS as a nanoscopic template. The hier-archically ordered structure of HMMS is successfully trans-ferred to the HMMP, as confirmed by the N2 isotherms andSAXS pattern (see Supporting Information). The pore-size

distribution of HMMP suggested 28-nm-sized mesocellularpores and 4-nm-sized ordered mesopores. The orderedstructure of HMMP with its two different length scales isalso clearly shown in the corresponding SAXS pattern. TheSAXS spectrum presents two types of scattering peaks indi-cated by two closed circles and one asterisk at q=0.18, 0.38,and 0.54 nm�1, respectively. The peaks indicated by a closedcircle represent cellular pores with a pore size of about35 nm. The peak indicated by the asterisk represents the or-dered small pores, which are generated by the negative rep-lication of ordered small pores of HMMS silica. The BETsurface area and single-point total pore volume of HMMPare 654 m2 g�1 and 1.49 cm3 g�1, respectively.

2.3. Application of HMMS Silica as a Host of EnzymeImmobilization

Since the development of stable enzyme systems is ofgreat importance for applications such as enzyme reactors,biosensors, and bioremediation, we have developed cross-linked enzyme aggregates (CLEAs) in HMMS, which canstabilize enzyme activity.[11,21] The preparation of CLEAs inHMMS requires a two-step process. The first step involvesthe adsorption of enzymes into HMMS, which proceedswith a high degree of enzyme loading within a short time.The second step involves glutaraldehyde (GA) treatment,which results in the crosslinking of enzyme molecules tocreate aggregates within the pores of HMMS. This approachis designed to yield stable enzyme activity by preventingleaching, since the enzyme aggregates created in the largermesocellular pores (37 nm) are not expected to leach outthrough the smaller mesoporous channels (13 nm). GAcrosslinking is performed promptly after the enzyme adsorp-tion, in order to enhance the high enzyme loading.

HMMS can be shown to be a good host material for thesynthesis of nanometer-sized enzyme aggregates for the fol-lowing reasons. First, 13-nm-sized connecting mesopores arelarge enough for the facile passage of individual enzymemolecules with little diffusional limitation. This results inthe quick adsorption of enzymes into HMMS. Second, themain 37-nm-sized spherical mesocellular pores can accom-modate nanometer-sized crosslinked enzyme aggregates,which cannot leach out of HMMS through smaller mesopo-rous channels. This would result in a stable enzyme systemas described below.

To demonstrate the concept of CLEAs in HMMS, weprepared CLEAs containing a-chymotrypsin (CLEA–CT).While shaking (200 rpm), CT adsorption with a high loadingof 36.3 wt % (570 mg of CT in 1 g of HMMS) was completedin less than five minutes. This rapid completion of highenzyme loading can be ascribed to both the small particlesizes and the good connectivity between the mesoporouschannels (13 nm) and large mesocellular pores (37 nm). CTadsorption was followed by GA treatment and excessivewashing for the preparation of CLEA–CT; the final CTloading in CLEA–CT was 33.2 wt%. In a control experi-ment without GA crosslinking, CT was leached from theHMMS during the incubation and washing steps, lowering

Figure 7. a) N2 adsorption/desorption isotherms of HMMC (Inset: Cor-responding pore-size distributions). b) Small-angle X-ray scatteringpattern of HMMC showing the regularity of the large cells and smallordered pores.




the final loading to only 6.8 wt%. This demonstrates thevalue of the GA treatment for capturing high enzyme load-ings.

Figure 8 shows the stability of free CT, adsorbed CT,and CLEA–CT in aqueous buffer (10 mm sodium phos-phate, pH 7.8) at room temperature. The samples wereshaken side-by-side (horizontally) at 200 rpm. These rigor-ous conditions for testing stability have not been used inmost other studies,[8,9] where the adsorbed enzymes in meso-

porous silica were claimed to be stable, though only beingincubated under static conditions. For practical applications,it is critical to preserve the initial activity under harsh condi-tions, since most reactions using immobilized enzymes areconducted under shaking. Adsorbed CT with no GA treat-ment showed a continuous loss of CT activity, and the leach-ing of CT was confirmed by measuring the amount of CT inthe supernatant. In contrast, CT activity was clearly stabi-lized when the samples were treated with 0.1% GA. ThisCLEA–CT showed no decrease in CT activity throughout atwo-week incubation period. The half-lives of free and ad-sorbed CT were calculated to be 1 h and 3.6 days, respec-tively. On the other hand, CLEA–CT did not show any ac-tivity loss even during extended incubation up to one month(data not shown). This impressive stability under rigorousconditions demonstrates that the “ship-in-a-bottle” ap-

proach was successful in preventing enzyme aggregates inthe main mesocellular pores (37 nm) from being leachedthrough the bottlenecks of the mesoporous channels(13 nm). We also speculate that most of the CLEA–CT inthe mesoporous channels are crosslinked with CLEAs inmesocellular pores and do not leach out from HMMS sincethe reduction of enzyme loading from adsorbed CT(36.3 wt %) to CLEA–CT (33.2 wt%) was marginal whencompared to the volume fraction of mesoporous channels.[22]

In addition, the observed stability indicates that CLEA–CTdoes not lose activity by autolysis due to the inhibition ofautolysis after the enzymes are crosslinked.

The kinetic constants of free CT, adsorbed CT, andCLEA–CT are shown in Table 1. The catalytic efficiency(kcat/Km) of CLEA–CT was 28 times lower than that of freeCT, and it was due to both reduced kcat (17 %) and in-creased Km (4.8 times) values. The reduced kcat value can beexplained by the reduced flexibility and deformation of CTenzyme molecules after being crosslinked. The increased Km

value can be attributed to the increased mass-transfer limi-tation for the substrate in HMMS containing CLEA–CT.However, since a soluble form of the free enzymes cannotbe recovered and recycled in real applications of enzymes,this lowered enzyme activity with CLEAs in HMMS is stilluseful in recycling enzymes and can be compensated by im-pressive stability and high enzyme loading, which makes itpossible to recycle enzymes for more iterative uses andreduce the size of enzyme reactors. In addition, the catalyticefficiency (kcat/Km) of CLEA–CT was 10 times higher thanthat of adsorbed CT due to higher kcat and lower Km valuesof CLEA–CT. This is an additional advantage of CLEA–CTover adsorbed CT, together with better stability and higherenzyme loading. We speculate that the higher kcat value ofCLEA–CT can be explained by the prevention of structuraldeformation of the CT molecules, which can be serious withadsorbed CT molecules due to denaturation and/or autoly-sis. Interestingly, adsorbed CT with lower CT loading(6.8 wt %) has a higher Km value than CLEA–CT withhigher loading (33.2 wt %). This suggests that adsorbed CTplaces more serious mass-transfer limitations on the sub-strate than CLEA–CT, even though the internal porosity ofHMMS with adsorbed CT contains smaller amounts of CTmolecules than that with CLEA–CT. This puzzling resultcan be explained by the denaturation and/or autolysis of ad-sorbed CT molecules, which leads to an increase of their oc-cupied volume and more serious mass-transfer limitation forsubstrates in HMMS. CLEA–CT would not have this kindof problem since multi-point attachments in the form of

CLEAs would prevent thedenaturation of CT mole-cules. We can even antici-pate a small degree ofvolume shrinkage duringcrosslinking and more con-trolled distribution ofCLEA–CT in HMMS. Inother words, adsorbed CTdoes not have any controlover the distribution of

Figure 8. Stability of free CT, adsorbed CT, and CLEA–CT under rigor-ous shaking (200 rpm). The relative activity (%) represents the ratioof residual activity to initial activity of each sample.

Table 1. CT loading and kinetic constants of free CT, adsorbed CT, and CLEA–CT.[a]

Samples CT Loading (wt%) kcat [s�1] Km [mm] kcat/Km [ � 103m�1 s�1]

Adsorbed CT 6.8 0.8�0.1 291�24 2.7�0.3CLEA–CT 33.2 5.2�0.4 186�23 28�4Free CT 29.9�0.7 39�3 770�6

[a] The CT activity was determined by the hydrolysis of TP (1.6–160 mm) in an aqueous buffer (10 mm

phosphate, pH 7.8) at room temperature (22 8C). The active-site concentrations were determined by theMUTMAC assay.[25] Kinetic constants were obtained by using software (Enzyme Kinetics Pro fromChemSW, Farifield, CA) that performs nonlinear regression based on the least-squares method.




enzyme molecules by nature, and small mesoporous chan-nels of HMMS may be continuously filled with intact, dena-tured, and autolyzed CT molecules, leading to a more detri-mental mass-transfer limitation than seen for CLEA–CT.

3. Conclusions

In conclusion, hierarchically ordered mesocellular meso-porous silica (HMMS) materials were synthesized using asingle structure-directing agent under neutral conditions. Asdescribed above, most of the hierarchical porous materialsreported so far are meso–macroporous or micro–macropo-rous materials. In addition, HMMS have two advantagesover other mesoporous materials. First, the overall syntheticprocess is very cost-effective because inexpensive sodiumsilicate was employed as the silica source and the synthesisis conducted under mild, neutral conditions. Secondly, thesynthetic procedure using a single template is much simplerthan those employed for the synthesis of other hierarchical-ly ordered mesoporous materials. Using HMMS as a nano-scopic template, hierarchically ordered mesocellular meso-porous carbon and polymer materials were successfully syn-thesized. Such silica, carbon, and polymer materials havethe potential to be used as a host of catalysts and withlarge-sized molecules such as biomolecules.

We also developed immobilized enzyme reactors in thenanometer-scale pores of a bimodal HMMS. Our approachusing glutaraldehyde for the purpose of crosslinking cap-tures very high enzyme loadings and prevents leaching, pro-viding a more stable and active immobilized enzyme systemthan those obtained by simple adsorption. Thus, we have de-veloped a “ship-in-a-bottle” approach to obtain active andstable enzyme reactors in the pores of a uniquely designedmesoporous material (HMMS). For example, CLEAs con-taining a-chymotrypsin (CLEA–CT) did not show any activ-ity decrease under rigorous shaking for one month, whichdemonstrates a huge success of this approach. This stableand active enzyme system is expected to make a broadimpact in various enzyme applications such as bioremedia-tion, biosensors, and bioconversion.

4. Experimental Section

Synthesis of hierarchically mesocellular mesoporous silica(HMMS): 9.7 g of P123 ((EO)20(PO)70(EO)20) and 4.48 mL of con-centrated acetic acid were dissolved in 200 mL of water. The re-sulting solution was heated to 60 8C and maintained at that tem-perature for 1 h. Then, 16 mL of sodium silicate diluted with200 mL of water was poured into the solution with vigorous stir-ring. On mixing the two solutions, the temperature was droppedto 45–47 8C. The pH value of the reaction mixture was 6.3–6.4.The molar composition of the synthetic mixture wasSiO2:P123:acetic acid:H2O =1:0.0167:0.078:222.2. The solutionwas reheated to 60 8C and aged at that temperature for 20 h, fol-

lowed by hydrothermal treatment at 100 8C for 24 h. Calcinationof the filtered materials at 550 8C generated HMMS.

Synthesis of HMMC: The alumination (Si/Al=20) of pure silicaHMMS was performed by means of the impregnation method, inorder to generate acidic catalytic sites. 1.3 mL phenol per gramof HMMS was incorporated into the pores of the HMMS, by heat-ing a mixture of HMMS and phenol at 140 8C under a staticvacuum. The resulting phenol-incorporated HMMS and formalde-hyde were reacted in an autoclave at 130 8C for 2 days insidethe pores of the HMMS to yield the phenol resin/HMMS nano-composite. The nanocomposite was heated to 160 8C at1 8C min�1 and held at this temperature for 5 h under flowing ni-trogen. The temperature was then ramped at 5 8C min�1 to 850 8Cand held at this temperature for 7 h to carbonize the phenolresin inside the pores of the HMMS, so as to obtain the carbon/HMMS nanocomposite. The dissolution of HMMS using 1 m

NaOH in a 1:1 mixture of EtOH and H2O generated HMMC.

Synthesis of HMMP: The calcined HMMS was dehydrated at200 8C under vacuum for 4 h. A polymer precursor solution com-posed of divinylbenzene and 2,2’-azobisisobutyronitrile (AIBN;15:1) was wetted into the pores of the HMMS silica using the in-cipient wetness method. The amount of divinylbenzene was ad-justed to 50 % of the pore volume of the HMMS template, inorder to preserve the cellular structure. Polymerization was per-formed by heating at 85 8C for 24 h under an argon atmosphere.Removal of the silica template using 10 wt % HF diluted with eth-anol yielded HMMP.

Enzyme immobilization in HMMS: HMMS (10 mg) was mixedwith 1.5 mL of 4 mg mL�1 free CT in a buffer solution (10 mm

sodium phosphate buffer, pH 7.8), vortexed for 30 s, sonicatedfor 3 s, and incubated at room temperature while shaking(200 rpm). After 20 min incubation for the adsorption of free CTin HMMS, the samples were washed very briefly in sodium phos-phate buffer (100 mm sodium phosphate, pH 8.0), and incubat-ed with 0.1 % glutaraldehyde solution in phosphate buffer(100 mm sodium phosphate buffer, pH 8.0) at 200 rpm for30 min. After GA treatment, the samples were washed by phos-phate buffer (100 mm sodium phosphate, pH 8.0) and Tris-HClbuffer (100 mm Tris, pH 8.0), respectively. The capping of un-reacted aldehyde groups was performed in a fresh Tris-HCl buffer(100 mm Tris, pH 8.0) at 200 rpm for 30 min. After Tris-capping,the samples were washed two times by phosphate buffer(10 mm sodium phosphate, pH 7.8), and stored at 4 8C. The ad-sorbed CT was also prepared by using no GA during the treat-ment process, but the washing was performed in the exactlysame way as for CLEA–CT. Protein leaching from HMMS into thesupernatant was measured by the BCA method[23] at each wash-ing step together with control samples, and used for the calcula-tion of final CT loading.

Activity and stability of CLEA–CT: The activity of the immobilizedCT was determined by the hydrolysis of 160 mm N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (TP) in an aqueous buffer (10 mm

sodium phosphate, pH 7.8) at room temperature. After 30 s ofvortexing, the samples were shaken at 250 rpm, and the in-crease in absorbance at 410 nm in the supernatant was mea-




sured time-dependently after centrifugation of the suspension at5000 G. The activity measurement was performed within 20 to30 min to reduce the possible effect of leached enzymes on theactivity results. The stability of CLEA–CT was checked in aqueousbuffer (10 mm sodium phosphate, pH 7.8) at room temperatureunder shaking (200 rpm). At each time point, an aliquot of eachsample was added to the aqueous buffer containing TP, and theresidual activity was measured as described above. The relativeactivity (%) was calculated from the ratio of the residual activityto the initial activity of each sample.

Characterization: Transmission electron microscopic images wereobtained on a JEOL EM-2010 microscope. Scanning electron mi-croscopic images were obtained on a JSM-840A microscope. N2

adsorption/desorption isotherms at 77 K were obtained using aMicromeritics ASAP2010 sorptometer. Pore-size distributionswere calculated using the BJH (Barett–Joyner–Halenda) method.Synchrotron SAXS measurements were performed on the 4C2Beamline at the Pohang Light Source (Korea). The primary beamwas monochromatized with a coupled Si(111) single crystal at awavelength of 0.1608 nm (the photon energy of the X-ray is7.78 keV, a resolution Dl/lffi0.0001), and then it was focusedon a detector plane by means of a bent cylindrical mirror. A 2DCCD camera (Roper Scientific Inc., PI-SCX-2048) was used to col-lect the scattered X-rays. We used the SEBS block copolymer(32.5 nm d spacing) as a periodic calibrant, in order to calibratethe image from the 2D CCD camera.[24]

Acknowledgments

T.H. is grateful for financial support from the Korean Ministryof Science and Technology through the National Creative Re-search Initiative Program. J.K. would like to thank U.S. Depart-ment of Energy (DOE) LDRD funds administered by the PacificNorthwest National Laboratory, and the DOE Office of Biologi-cal and Environmental Research under the EnvironmentalManagement Science Program. The research was performedin part at the W. R. Wiley Environmental Molecular SciencesLaboratory, a national scientific user facility sponsored by theU.S. Department of Energy’s Office of Biological and Environ-mental Research and located at Pacific Northwest NationalLaboratory. We thank Prof. Chae-Ho Shin at the Chungbuk Na-tional University for the micropore structure characterization.We also thank Dr. Hyunmin Park at the Korea Research Insti-tute of Standards and Science for the small-angle X-ray scat-tering studies.

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Received: January 27, 2005Published online on May 11, 2005




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